The goal of this project is to understand the mechanisms underlying remodeling of cardiac t-tubules in response to various stresses associated with cardiac disorders. T-tubules are extensions of sarcolemmal membrane harboring essential elements involved in excitation-contraction coupling. While t-tubules appear to be constant features of adult ventricular myocytes under normal conditions they become disorganized or even lost during a number of pathologies including heart failure. However, despite finding strong correlation between t-tubular remodeling and changes in t-tubular structural proteins, it remains essentially unknown why and how t-tubular remodeling occurs. We have recently shown that t-tubules become constricted or even sealed off in response to removal, but not induction, of membrane stretch caused by brief hyposmotic shock. Preliminary data show that similar t-tubular remodeling also occurs in intact hearts after ischemia-reperfusion challenge which is associated with cell swelling and membrane stretch. Accordingly, this project will test an overall hypothesis that acute membrane stretch induced during various stress conditions leads to disruption of t- tubular system and consequent cardiac dysfunction. Aim 1 will determine molecular and cellular mechanisms underlying t-tubular remodeling in response to various acute stresses using a combination of 'state of the art' established approaches such as time-lapse 3D confocal microscopy as well as novel technology of electrophysiological recordings from whole individual t-tubules assisted by super-resolution Scanning Ion Conductance Microscopy. Aim 2 will determine the cellular consequences of stress-induced t-tubular remodeling. It will provide detailed functional assessment of remodeled t-tubular system, including characterization of ionic homeostasis of sealed off t-tubules. In particular, this Aim will test a novel hypothesis that partially sealed t-tubules provide an unconventional pathway of Ca2+ entry into myocytes. Aim 3 will test a hypothesis that t-tubule sealing is a common mechanism leading to disruption of excitation-contraction coupling, cardiomyopathy and heart failure. Here, we will employ an isolated mouse heart model to get mechanistic insights into the origins of cardiac dysfunction in response to acute osmotic stress, pressure overload and ischemia reperfusion challenge. Overall, results of this project will contribute significantly to better understanding of the origins of various stress-related disorders and thus will help in development of novel approaches in the treatment and prevention of relevant cardiac diseases.